Changes in microbial dynamics during vermicomposting of fresh and composted sewage sludge

Waste Management xxx (2015) xxx–xxx

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Changes in microbial dynamics during vermicomposting of fresh and composted sewage sludge Iria Villar ⇑, David Alves, Domingo Pérez-Díaz, Salustiano Mato Department of Ecology and Animal Biology, University of Vigo, 36310 Vigo, Spain

a r t i c l e

i n f o

Article history: Received 7 May 2015 Revised 7 October 2015 Accepted 8 October 2015 Available online xxxx Keywords: Enzyme activities PLFAs Microbial community Earthworm Organic waste Stability

a b s t r a c t Municipal sewage sludge is a waste with high organic load generated in large quantities that can be treated by biodegradation techniques to reduce its risk to the environment. This research studies vermicomposting and vermicomposting after composting of sewage sludge with the earthworm specie Eisenia andrei. In order to determine the effect that earthworms cause on the microbial dynamics depending on the treatment, the structure and activity of the microbial community was assessed using phospholipid fatty acid analysis and enzyme activities, during 112 days of vermicomposting of fresh and composted sewage sludge, with and without earthworms. The presence of earthworms significantly reduced microbial biomass and all microbial groups (Gram+ bacteria, Gram bacteria and fungi), as well as cellulase and alkaline phosphatase activities. Combined composting–vermicomposting treatment showed a lesser development of earthworms, higher bacterial and fungal biomass than vermicomposting treatment and greater differences, compared with the control without earthworms, in cellulase, b-glucosidase, alkaline and acid phosphatase. Both treatments were suitable for the stabilization of municipal sewage sludge and the combined composting–vermicomposting treatment can be a viable process for maturation of fresh compost. Ó 2015 Published by Elsevier Ltd.

1. Introduction Municipal wastewater treatment plants produce significant amounts of sewage sludge, amount to about 11 million dry tonnes per year in the EU, which needs suitable and environmentally accepted management before final disposal (Kelessidis and Stasinakis, 2012). Sewage sludge can harm the environment when it is deposited directly on soil due to its fermentative capacity and the presence of hazardous substances, both organic and inorganic, including pathogenic organisms and heavy metals (Williams, 2005). Due to its high organic load, sewage sludge is a suitable waste for being treated by biological techniques such as composting and vermicomposting aimed at obtaining a stable product with a high agronomic value. Vermicomposting is a bio-oxidation and stabilization process of organic matter as a result of the interaction between microorganisms and earthworms. Microorganisms are mainly responsible for the degradation of organic material, although earthworms stimulate microorganisms due to the modification of substrate properties through feeding, aeration and cast excretion, which ⇑ Corresponding author. E-mail address: [email protected] (I. Villar).

leads to the acceleration of mineralisation of organic matter and the improvement of nutrient availability for plants (Domínguez, 2004). Vermicomposting has been successfully applied on the treatment of municipal sewage sludge. Most research on sewage sludge vermicomposting has focused on the study of physical– chemical parameters such as nutrients (Domínguez and GómezBrandón, 2013; Fu et al., 2015), humic and fulvic substances (Zhang et al., 2015) and heavy metals (Suthar, 2010). Nevertheless, less is reported on the microbiological and biochemical changes that occur during the vermicomposting of municipal sewage sludge. Benitez et al. (1999) observed a reduction in bglucosidase, protease, urease and dehydrogenase activities related to the decline in available substrates, in the first 6 weeks of vermicomposting of municipal sewage sludge mixed with paper mill sewage sludge. Domínguez and Gómez-Brandón (2013) found that the presence of the earthworm Eisenia andrei increased microbial biomass, measured as N-microbial biomass by fumigationextraction method, from week 1 to week 16 of vermicomposting of sewage sludge compared to the control without earthworms. On the contrary, Fu et al. (2015) observed a decrease of C-microbial biomass in the first 40 days of vermicomposting of pelletized dewatered sludge, with subsequent low and constant values that indicated the stability of the final products.

http://dx.doi.org/10.1016/j.wasman.2015.10.011 0956-053X/Ó 2015 Published by Elsevier Ltd.

Please cite this article in press as: Villar, I., et al. Changes in microbial dynamics during vermicomposting of fresh and composted sewage sludge. Waste Management (2015), http://dx.doi.org/10.1016/j.wasman.2015.10.011

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The integration of composting and vermicomposting has been considered a suitable method for waste management (Ndegwa and Thompson, 2001). The inoculation of earthworms in the material that passed through the thermophilic phase of composting has been used as a pre-treatment before vermicomposting, in order to remove compounds harmful for earthworms, such as ammonium (Domínguez, 2004). Several authors have investigated the combined use of composting and vermicomposting for the treatment of different organic materials, showing that prior composting can accelerate degradation and improve the stabilization of the final product (Frederickson et al., 1997; Lazcano et al., 2008). Fornes et al. (2012) studied the evolution of composting, vermicomposting and the combined composting–vermicomposting process of horticulture waste, focusing their research on the physical–chemical changes over time. These authors showed that vermicomposts had better properties as growing media than as compost. Also, Lazcano et al. (2008) compared the products of these processes, but not the evolution over time, by the study of microbiological and biochemical parameters, noting that the combined process was the most effective method for the stabilization of the cattle manure. Hait and Tare (2011) reported that the combined compost ing–vermicomposting process of sewage sludge made it possible to obtain a good quality pathogen-free product. Likewise, Sen and Chandra (2009) showed that earthworms changed the dynamic of the bacterial community in the combined process compared with composting of sugar waste, but they did not study the vermicomposting process. So, no research on microbiological evolution over time of the vermicomposting process and the combined composting–vermicomposting process for the same waste was found. It has been observed that the study of enzyme activities is a reliable index of the evolution of organic matter during vermicomposting (Benitez et al., 1999; Aira et al., 2007a). Enzyme activities provide information on the conversion of complex organic compounds into more readily assimilable substances and, hence, enzymes are of interest to evaluate stabilization throughout waste biodegradation. Likewise, enzyme activities have been related to earthworm growth. Thus, they have been proposed as indicators to optimise vermicomposting process (Fernández-Gómez et al., 2010). Benitez et al. (1999) demonstrated that hydrolytic enzyme activities tended towards stability in the course of sewage sludge vermicomposting, but however, the lack of controls makes it difficult to distinguish between the effect caused by earthworms and the effect of the microbiota present in the waste. Conversely, phospholipid fatty acid analysis (PLFAs) is a useful tool for monitoring the microbial community dynamics. The total amount of PLFAs can be used as an indicator of viable microbial biomass and some PLFAs are specific to certain living organisms and, therefore, can be used as biomarkers for the presence and abundance of microbial groups (Zelles, 1999). Thus, analysing PLFAs during vermicomposting makes it possible to know the changes in the microbial community composition over time. Fernández-Gómez et al. (2013) observed a reduction in total PLFAs of different organic wastes after 24 weeks of vermicomposting. In the same way, Gómez-Brandón et al. (2011a, 2013) reported that the activity of earthworms reduced the PLFAs characteristic of bacterial and fungal biomass. This reduction was more pronounced between week 21 and week 36 of rabbit manure vermicomposting and pig slurry vermicomposting. In this work, we studied the microbiological evolution during vermicomposting compared with vermicomposting after composting, for the treatment and stabilization of sewage sludge. The main hypothesis was that earthworms cause a different effect on microbial community structure, depending on whether they feed on fresh or previously composted material. To this end, enzyme activities (cellulase, b-glucosidase, protease, alkaline and acid

phosphatase) and the structure of the microbial community by analysing PLFAs were assessed throughout the vermicomposting of fresh and composted sewage sludge with the earthworm specie E. andrei. In order to discern the effects due to the different microbiological composition of the waste from the effects caused by earthworms, the same substrates incubated without earthworms were studied. 2. Materials and methods 2.1. Substrates and earthworms Sewage sludge was collected from a municipal wastewater treatment plant in Cangas (Pontevedra, NW Spain) after an aerobic biological treatment and subsequent dehydration. The sludge was mixed with wood chips as a bulking agent, adjusting the ratio to 1:2 (v/v). A part of this mixture was used for the vermicomposting treatment (V). Another part of the mixture was subjected to composting in a static adiabatic reactor with a 600 L capacity and automatic control of temperature and oxygen. Forced aeration was applied, using a centrifugal fan intermittently and depending on the controlled variables. The temperature was maintained above 45 °C for 7 days with maximum values of 60 °C. The process ended after 15 days when the temperature in the composting mass reached values below 35 °C. The fresh compost was removed from the reactor, mixed and used as a substrate for vermicomposting in the combined composting–vermicomposting treatment (CV). The earthworm species E. andrei was used for the vermicomposting due to its high tolerance to environmental factors and its high rate of organic matter processing (Domínguez, 2004). In order to determine if the substrates affected the growth and maturation of the earthworms, juvenile specimens with an average weight of 310 ± 25 mg were collected from a laboratory culture fed with horse manure. 2.2. Experimental design Vermicomposting was carried out in rectangular culture systems of 14 L capacity, which were filled with a layer of sieved and moistened vermiculite as refuge for earthworms, with the advantage of being a biologically inert material. A plastic mesh (5 cm mesh size) was placed between the vermiculite and the substrate to prevent their mixture and facilitate the sampling. Two kilograms of substrate sludge or compost (2000 ± 6 g) and 115–120 earthworms according to the feed rate of 0.75 kg feed/kg worm/day (Ndegwa et al., 2000), were introduced. Each substrate was replicated three times. Controls involving the same materials (vermiculite, mesh and sludge or compost) incubated without earthworms were included in triplicate. Culture systems were kept in darkness under the same conditions. The moisture content was controlled and maintained above 70% by watering throughout the process. After 70 days, cocoons, earthworms and hatchlings were removed by hand from the cultures, counted and weighed. The culture systems were maintained until day 112 to enable the maturation of the vermicompost. Samples were taken at 0, 14, 28, 42, 56, 70, 91 and 112 days. In order to remove the bulking agent, samples were sieved (less than 10 mm) and several parameters were determined, as detailed below. 2.3. Physical–chemical analysis Organic matter content was measured by the loss on ignition of dried samples at 550 °C for 4 h. Inorganic nitrogen (N-NH+4 and N-NO3 ) was determined in 0.5 M K2SO4 extracts in a ratio of 1:10 (w/v) applying the modified indophenol blue colorimetric

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method (Sims et al., 1995). Total extractable nitrogen was determined in the same extracts after oxidation with K2S2O8, as described by Cabrera and Beare (1993), and dissolved organic nitrogen content (DON) was calculated as (total extractable N) – (inorganic N). Total nitrogen content (TN) and total carbon content (TC) were determined by combustion of dried samples using a LECO 2000 CN elemental analyser. Water soluble carbon content (WSC) was analysed in aqueous extracts 1:5 (w/v) after oxidation with H2SO4 (96%) and K2Cr2O7 (1N) at 160 °C for 30 min and spectrophotometric measurement of reduced chromium. The pH was determined in aqueous extracts 1:10 (w/v) using a pH metre Crison Basic 20.

t-tests were performed to determine the difference between the two types of materials used in vermicomposting and one-way analysis of variance (ANOVA) to determine the difference between physical–chemical parameters at the end of the vermicomposting process. For post hoc comparison between groups in the case of a significant effect, HSD Tukey tests were used. Where the assumptions of normality and variance homogeneity were not met, data were log-transformed. All statistical tests were evaluated at the 95% confidence level using the SPSS 20.0 programme. 3. Results and discussion 3.1. Growth and reproduction of E. andrei

2.4. Biological and biochemical analysis b-Glucosidase was estimated by incubating the sample (1 g fresh weight) with 1 mL of p-nitrophenyl-b-D-glucopyranoside (0.025 M) for 1 h at 37 °C and subsequent colorimetric measurement of p-nitrophenol released (Eivazi and Tabatabai, 1988). Alkaline and acid phosphatase was measured by incubating the sample (0.5 g fresh weight) with 1 mL of p-nitrophenylphosphate (0.015 M) for 1 h at 37 °C and subsequent colorimetric measurement of p-nitrophenol released (Eivazi and Tabatabai, 1977). Protease activity was measured by colorimetric determination of the amino acids released, after the incubation of the sample (1 g fresh weight) with 5 mL of sodium caseinate (2%) for 2 h at 50 °C, using Folin–Ciocalteu reagent (Ladd and Butler, 1972). Cellulase activity was assessed by colorimetric determination of reducing sugars released after incubation of the sample (5 g fresh weight) with 15 mL of carboxymethyl cellulose sodium salt (0.7%) for 24 h at 50 °C (Schinner and Von Mersi, 1990). Germination index (GI) was calculated according to Zucconi et al. (1981) by determining seed germination and root length of Lepidium sativum growing in 2 mL of aqueous extracts 1:5 (w/v) in Petri dishes lined with paper filter during 48 h. The microbial community composition and biomass was determined by phospholipid fatty acid analysis (PLFAs) following the method described by Gómez-Brandón et al. (2010) for organic samples. Briefly, total lipids were extracted by stirring from 200 mg of each freeze-dried sample with 60 mL of chloroform– methanol (2:1, v/v) and separated into neutral lipids, glycolipids and phospholipids on silicic acid columns. The phospholipid fraction was subjected to derivatization with trimethylsulfonium hydroxide (TMSH) and fatty acid methyl esters (FAMEs) obtained were analysed by gas chromatography and mass spectrometry (GC–MS). GC–MS analysis was performed on a column CP-Select FAME, 100 m  0.25 mm. FAMEs were identified by comparison of their retention time and mass spectra with known standards (Larodan Fine Chemicals AB, Malmo, Sweden). The quantification was performed using internal standard calibration. PLFAs were used to estimate the biomass of specific microbial groups: grampositive bacteria (i14:0, i15:0, a15:0, i16:0, a17:0), gram-negative bacteria (16:1x7, cy17:0, 17:1x7, 18:1x7, cy19:0) and fungi (18:2x6, 18:1x9, 20:1x9) (Frostegård and Bååth, 1996; Zelles, 1997; Madan et al., 2002). The total amount of PLFAs identified (totPLFAs) was used as an indicator of the viable microbial biomass (Zelles, 1999).

As shown in Table 1, the survival of E. andrei in both treatments V and CV was high (>96.5% in all culture systems) and no significant differences were found (p > 0.05), so fresh and composted sewage sludge presented good properties for their management by vermicomposting. The growth of E. andrei in V treatment was significantly larger than in VC treatment (t = 9.063, p < 0.05) and earthworm biomass significantly increased relative to the beginning of the process, about 51.3% in V (t = 16.206, p < 0.01) and 25.6% in CV (t = 14.042, p < 0.01). The highest number of mature earthworms, cocoons and hatchlings were obtained in V treatment, with significant differences between treatments (t = 4.706, p < 0.05; t = 9.347, p < 0.05; t = 16.818, p < 0.05, respectively). These results suggested that the vermicomposting of sewage sludge showed slightly better conditions for the development of E. andrei than composted sludge. Frederickson et al. (1997) found that the growth rate of the epigeic earthworm Eisenia fetida was reduced in pre-composted green waste compared with vermicomposting of fresh material, suggesting that the nutritional content was more rapidly decreased during the early stages of composting. Gunadi and Edwards (2003) propounded that pre-composting of cattle manure can reduce bioavailable nutrients for earthworms, inhibiting the growth rate and the number of cocoons and hatchlings produced by E. fetida. Similar results were established in this study for municipal wastewater sewage sludge. Moreover, CV treatment presented greater concentrations of harmful compounds for earthworms, such as ammonium (Table 2), which could negatively affect earthworm development. 3.2. Physical–chemical parameters The physical–chemical properties at the initial and final materials are shown in Table 2. The organic matter content was significantly lower in the presence of earthworms than in controls (F1,11 = 62.483, p < 0.0001). After vermicomposting, organic matter decreased about 14.6% in V treatment with earthworms and 3.4% in treatment without earthworms, while reductions were about 12.7% and 6.6% in CV treatment with and without earthworms,

Table 1 Growth, sexual development and survival of E. andrei after 70 days of vermicomposting (V) and composting–vermicomposting (CV). Values are mean ± standard error (n = 3).

2.5. Statistical analysis Microbiological and biochemical data were analysed by repeated measures analysis of variance (ANOVAR) in which the type of material and the presence or absence of earthworms were set as between-subjects factors, and time was set as withinsubjects factor. Correlation analyses were carried out to examine the relationships between PLFAs and enzyme activities. Student’s

Earthworm biomass (g) Weight gain per earthworm (mg) Matured earthworms (%) No. of hatchlings No. of cocoons Survival (%)

V

CV

54.8 ± 1.2a 169.4 ± 5.1a 95.0 ± 1.3a 517 ± 54a 840 ± 42a 97.7 ± 1.1a

45.3 ± 0.6b 80.7 ± 6.2b 86.3 ± 2.3b 10 ± 1b 282 ± 11b 99.4 ± 0.3a

Means with the same letter are not significantly different (paired-sample Student’s t-test, p < 0.05).

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Table 2 Initial and final physical–chemical properties of vermicomposting (V) and composting–vermicomposting (CV). Values are means ± standard error (n = 3). Time OM (g)

0 112

pH

0 112

C to N ratio TC (g kg TN (g kg

1

1

WSC (g kg DON (g kg NH+4

(g kg

GI (%)

0 112

dw) dw) 1

1

1

dw) dw)

dw)

0 112 0 112 0 112 0 112 0 112 0 112

V_E.andrei

V_control

CV_E.andrei

a

495.0 ± 1.1 421.2 ± 1.0a

507.4 ± 0.5 480.2 ± 0.7b

6.2 ± 0.1

443.5 ± 1.1c

a

5.6 ± 0.0a

401.8 ± 1.6 350.1 ± 7.6b

323.4 ± 5.5a

39.0 ± 0.6a 33.4 ± 0.5a

30.7 ± 0.4b

31.2 ± 1.0ab b

2.64 ± 0.13

7.53 ± 0.33 4.43 ± 0.10b

3.02 ± 0.10a

a

3.42 ± 0.05c b

4.57 ± 0.08

5.16 ± 0.15 7.88 ± 0.22b

3.84 ± 0.23c

a

3.95 ± 0.13c b

0.69 ± 0.04 0.20 ± 0.01a

349.2 ± 5.1b 38.9 ± 0.3a

a

6.56 ± 0.08a

11.2 ± 0.2b b

428.0 ± 1.9

2.88 ± 0.11a

a

10.5 ± 0.3ab

a

32.3 ± 0.2ab

6.0 ± 0.1b 9.6 ± 0.1

10.5 ± 0.3ab

319.6 ± 7.1a

b

5.9 ± 0.0b

a

9.9 ± 0.3a

2.41 ± 0.14 0.37 ± 0.03b

0.28 ± 0.02c

65.8 ± 2.3a 92.1 ± 0.4a

473.1 ± 4.1b 7.1 ± 0.0

5.7 ± 0.0a 10.2 ± 0.2

CV_control b

0.37 ± 0.02b 33.3 ± 1.1b

76.7 ± 0.9b

97.9 ± 0.4c

86.2 ± 0.5d

OM: organic matter, dw: dry weight, TC: total carbon, TN: total nitrogen, WSC: water soluble carbon, DON: dissolved organic nitrogen, GI: germination index. Means with different letter in the same row are significantly different (Tukey HSD, p < 0.05).

respectively. The presence of earthworms accelerated the mineralisation of organic matter (Elvira et al., 1996) so that the highest earthworm biomass in V treatment may have produced a greater decrease in this measure. A greater reduction was noted in organic matter in VC controls compared to V controls, which showed a higher loss of organic matter through microbial breaking down after the passage of sewage sludge at the thermophilic phase of composting. Ryckeboer et al. (2003) pointed out that the taxonomic and metabolic diversity of bacteria and available substrates for fungi increase after the thermophilic phase of composting. All final products had acidic conditions and there were no significant differences between the presence and absence of earthworms, but V treatment showed lower pH than CV (F1,11 = 24.666, p < 0.0001). These results agreed with those obtained by other authors (Ndegwa et al., 2000; Khwairakpam and Bhargava, 2009; Hait and Tare, 2011), who observed a decrease in pH after vermicomposting of sewage sludge due to the formation of organic acidic compounds and the mineralisation of nitrogen and phosphorus. The decrease in carbon and nitrogen content, due to the degradation and mineralisation of organic matter by microorganisms, earthworms and the joint action of both, maintained the C–N ratio at a low level in all treatments, with similar initial and final values. This ratio is extensively used as an indicator of maturity of organic waste, although Yadav et al. (2010) reported that the C–N ratio should not be used as a maturity parameter for the vermicomposting if the original waste is rich in nitrogen. A significant reduction of TN and TC was observed between initial and final values with significant differences between all treatments (F3,11 = 153.409, p < 0.0001). With respect to TN, it was reduced by 21% on average in CV and 16% on average in V, finding significant differences between V and CV treatments (F1,11 = 10.935, p < 0.01). The low C–N ratio in the substrates and the aeration provided by the bulking agent may cause the release of ammonia gas and decrease of TN in vermicomposting (Benitez et al., 1999; Domínguez, 2004). In the case of DON, significant differences between V and CV (F1,11 = 98.193, p 6 0.0001) were detected with a reduction in CV and an increase in V in both treatments with earthworms and controls. The presence of E. andrei showed lower concentrations of ammonium (F1,11 = 0.437, p < 0.01), WSC (F1,11 = 16.131, p < 0.01) and CT (F1,11 = 23.478, p < 0.01) than controls. These results were

consistent with the general hypothesis that earthworms promote carbon and nitrogen mineralisation (Domínguez, 2004). Although readily assimilable carbon and nitrogen content increased after composting, the subsequent process of vermicomposting diminished these parameters, favouring the stabilization of combined CV treatment. Also, the presence of E. andrei showed significantly higher GI than controls (F1,11 = 21.023, p < 0.01) and the CV treatment presented the greatest value (97.9%). According to Zucconi et al. (1985), values of germination index greater than 80% present no phytotoxicity, so that both treatments with earthworms were effective for eliminating phytotoxic substances, suggesting a suitable level of maturation. The physical–chemical parameters evaluated and the germination index showed that earthworms improved the sewage sludge properties, reaching optimal values of stabilization and maturation in both V and CV treatments. 3.3. Enzyme activities In general, the five hydrolytic enzymes studied decreased throughout all treatments (Figs. 1 and 2) accordingly, with metabolic degradation processes of organic matter diminishing during vermicomposting. Benitez et al. (1999) observed similar trends during vermicomposting of sewage sludge, suggesting that the decrease in hydrolytic activities indicated stabilization of organic matter. Cellulase and b-glucosidase are enzymes of the carbon cycle that play an important role in the breakdown of organic matter. Cellulases degrade cellulose, releasing reducing sugars and b-glucosidases catalyse the hydrolysis of b-glycosidic bonds of the carbohydrates (Alef and Nannipieri, 1995). The cellulase activity decreased after composting and significant differences were observed between V and CV, both at the outset (t = 4.455, p < 0.05) and during vermicomposting (F1,8 = 8.607, p < 0.05) (Fig. 1a and b). Owing to cellulose being broken down by thermophilic organisms (Ryckeboer et al., 2003), the decrease in substrates after composting may have reduced cellulase activity. In V treatment, with and without earthworms, cellulase activity diminished in a similar way over the time, whereas in CV treatment the decline was clearly higher in the presence of earthworms compared to controls from day 28. Earthworms significantly

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Fig. 1. Changes in cellulase and b-glucosidase activities with and without E. andrei in vermicomposting (V) (a and c) and composting–vermicomposting (CV) (b and d). Values are mean ± standard error (n = 3).

reduced cellulase activity (F1,8 = 40.530, p < 0.0001) producing a significant interaction between treatment, time and earthworms presence/absence (F6,48 = 4.302, p < 0.01). Therefore, not only the presence of E. andrei resulted in a decrease of cellulase activity, but also the effect caused by time and the type of substrate, that probably affected the available substrate for this enzyme and cellulolytic microbial community. Due to the fact that earthworms can feed on fungi (Schönholzer et al., 1999), the main consumers of cellulose, changes in fungal community by earthworms, as has been observed in this study by the analysis of PLFAs, could affect the production of cellulases. Likewise, Gómez-Brandón et al. (2011b) found that earthworm activity reduced cellulase enzyme after vermicomposting of grape marc, suggesting that the vermicomposted material reached a high degree of stabilization. Regarding b-glucosidase, no significant differences between V and CV were detected at the beginning of vermicomposting (t = 0.251, p > 0.05), these being about 4200 lg PNP g 1 dw h 1 (Fig. 1c and d). b-Glucosidase activity presented significant differences between V and CV during vermicomposting (F1,8 = 36.319, p < 0.0001), producing a significant interaction between treatment, time and earthworms presence/absence (F6,48 = 5.374, p < 0.0001). There was a gradual decline in enzyme b-glucosidase in V treatment with a minimum at the end of the experiment (85% reduction), both in the presence and absence of earthworms. In the CV treatment, the decline in the first sampling involved about 60% reduction of the enzyme activity, with and without earthworms, and then the activity remained stable from 56 days. Similar results were obtained by Benitez et al. (1999) during vermicomposting of sewage sludge, showing a sharp decrease in b-glucosidase activity during the first 6 weeks with a subsequent stabilization trend as a result of the decrease in available organic substrates. Moreover, when the availability of C and N required for enzyme synthesis is limiting, microorganisms can constrain their production of enzymes (Allison and Vitousek, 2005). Sewage sludge showed a low C–N ratio (Table 2), which may lead to carbon becoming a

limiting substrate for the microbiota. These results showed differences between treatments for enzymes of the C cycle, indicating that enzyme activities decreased and stabilized before in combined composting–vermicomposting than in vermicomposting of fresh sewage sludge. Alkaline and acid phosphatase enzymes catalyse the hydrolysis of organic phosphomonoester to inorganic phosphorus, differing according to their optimum pH of activity. At the beginning of vermicomposting, significant differences were observed between V and CV for both acid phosphatase (t = 7045, p < 0.05) (Fig. 2a and b) and for alkaline (t = 3442, p < 0.05) (Fig. 2c and d). Differences in the activities were also detected between the two treatments for the two enzymes throughout the process (F1,8 = 7.147, p < 0.05 for acid phosphatase; F1,8 = 6.153, p < 0.05 for alkaline phosphatase). Phosphatase activities decreased with time, but because phosphatases are highly influenced by the pH (Eivazi and Tabatabai, 1977), a further decrease of the alkaline phosphatase with values greater than 60% reduction was observed. The accumulation of inorganic compounds of phosphorus as a result of enzymatic activity has been shown to repress phosphatase activity (Alef and Nannipieri, 1995). Likewise, Aira et al. (2007a) suggested that a decrease in microbial biomass may cause an increase of available phosphorus and, therefore, a decrease in phosphatase activity. This drop in phosphatase activity was also observed by other authors in the vermicomposting of different wastes (Fernández-Gómez et al., 2010, 2013). In V treatment (Fig. 2a and c), phosphatase activities evolved in a similar way, regardless of the presence or absence of earthworms. However, a greater influence of E. andrei on enzyme activity was observed in the CV treatment (Fig. 2b and d) with a faster decrease in phosphatase activities in the presence of earthworms, resulting in significant interaction between treatment, time and earthworms presence/absence in acid (F6,48 = 10.810, p < 0.0001) and alkaline phosphatase (F6,48 = 1.961, p < 0.05). Furthermore, earthworms significantly affected alkaline phosphatase (F1,8 = 24.109, p < 0.01).

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Fig. 2. Changes in acid phosphatase, alkaline phosphatase and protease activities with and without E. andrei in vermicomposting (V) (a, c, and e) and composting– vermicomposting (CV) (b, d, and f). Values are mean ± standard error (n = 3).

Earthworms had a greater effect on the enzymes of the phosphorus cycle in the combined CV treatment than V treatment. Microbiota decrease and mineralisation increase during composting, with subsequent activity of earthworms, may have increased the content of orthophosphate and reduced the activity and synthesis of phosphatase in CV treatment. Protease enzymes supply a large part of the available nitrogen by catalysing the hydrolysis of proteins to peptides and amino acids (Alef and Nannipieri, 1995; Geisseler and Horwath, 2008). Significant differences between V and CV (t = 11.046, p < 0.01) were detected at the beginning of vermicomposting (Fig. 2e and f). Protease activity decreased in all treatments with a greater reduction in V compared to CV (F1,8 = 36.986, p < 0.0001). Protease activity is dependent on substrate availability (Aira et al., 2007a), which was reduced as the vermicomposting process developed. Also, the greater activity in the final samples of CV treatment could be caused by the formation of complexes between extracellular enzymes and humic substances because of the previous process of composting that can increase the formation of these substances compared to vermicomposting (Campitelli and Ceppi, 2008). The evolution through time in V treatment was

similar with and without earthworms, showing a decrease in the first 14 days of process, remaining stable up to 70 days and decreasing around 5000 lg tyrosine g 1 dw 2 h 1 in the final sampling. In CV treatment without earthworms, protease activity slightly increased over time until it reduced from 70 days to similar values to those observed at the beginning of the process. In the case of CV treatment with earthworms, two different stages were observed: a first stage where activity increased to a maximum at 28 days (17,700 lg tyrosine g 1 dw 2 h 1) and a second stage, from 42 days to 112 days, where the activity fell below the values noted at the beginning of the process. No significant differences between treatments with earthworms and controls were detected, although there was a significant interaction between treatment, time and earthworms presence/absence (F6,48 = 9.273, p < 0.0001). In general, a reduction in protease was observed over time, more marked in the treatment V and with a slight negative effect of earthworms on this enzymatic activity. Several studies have observed reductions of protease activity in the presence of earthworms in contrast to controls with different organic substrates (Aira et al., 2007a; Gómez-Brandón et al., 2011b; Fernández-Gómez et al., 2013). Geisseler and Horwath (2008) suggested that microorganisms

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7

Fig. 3. Changes in microbial biomass, measured as totPLFAs, with and without E. andrei in vermicomposting (V) and composting–vermicomposting (CV). Bars represent standard errors. Different letters in the same time of sampling are significantly different (Tukey post hoc test p < 0.05).

regulate protease synthesis, depending on their needs of carbon and nitrogen, so that the low C to N ratio could cause a decrease in microbial biomass and enzyme synthesis. 3.4. Dynamic of the microbial community Microbial biomass presented significant differences between V and CV (t = 21.719, p < 0.01) at the beginning of vermicomposting with a totPLFAs content in the first case of 1280 lg g 1 dw, and after the composting process this was reduced up to 895 lg g 1 dw. Both treatments showed a decrease in microbial biomass throughout the vermicomposting process (Fig. 3), especially in the first samplings, resulting in a significant interaction between treatment and time (F6,48 = 70.315, p < 0.0001). This decline in microbial biomass, measured as totPLFAs, was consistent with the results obtained by other authors during vermicomposting (Fernández-Gómez et al., 2013; Gómez-Brandón et al., 2013). The sewage sludge used in this experiment was biologically degraded during processing at the plant, so that the easily degradable nutrients could exhaust, thereby resulting in a reduction of microbial biomass. Earthworm activity greatly reduced the abundance of totPLFAs (F1,8 = 117.789, p < 0.0001), although this effect was more pronounced in V treatment (98.5% reduction from initial) compared with CV (89.4% reduction from initial), producing a significant interaction between treatment and presence /absence of earthworms (F1,8 = 6.469, p < 0.05). Because V treatment presented a higher increase in earthworm population and biomass compared to that observed in CV treatment, microbial biomass showed a larger decline in totPLFAs. These findings are consistent with previous observations that suggest that the digestion of organic material by epigeic earthworms has negative effects on microbial biomass (Gómez-Brandón et al., 2011c). In addition, Tiunov and Scheu (2004) observed that earthworms can compete with microorganisms for available carbon resources and cause a sharp decline in microbial biomass when carbon is limiting. The reduction of available resources due to the action of microorganisms, competition for them with earthworms and feeding with bacteria and fungi by earthworms, may explain the higher decline in biomass in the presence of E. andrei. In V treatment with earthworms, totPLFAs correlated positively with all enzymes: cellulase (r = 0.787, p < 0.0001), alkaline phosphatase (r = 0.832, p < 0.0001), b-glucosidase (r = 0.942, p < 0.0001), acid phosphatase (r = 0.684, p < 0.001) and protease (r = 0.750, p < 0.0001), with a

slightly lower correlation in all enzymes (p < 0.01) in absence of earthworms. In CV treatment with earthworms, correlation between totPLFAs with cellulase (r = 0.791, p < 0.0001), glucosidase (r = 0.649, p < 0.01), protease (r = 0.481, p < 0.05), alkaline (r = 0.822, p < 0.0001) and acid phosphatase (r = 0.601, p < 0.001) were observed. No correlations were found, however, between totPLFAs and acid phosphatase and protease in CV control, whereas correlations were detected with cellulase (r = 0.533, p < 0.01), alkaline phosphatase (r = 0.763, p < 0.01) and b-glucosidase (r = 642, p < 0.01). Despite the different evolution of hydrolytic enzymes, the results in general showed that a decrease in microbial biomass was accompanied by a decrease in enzymes, suggesting that enzyme activities were directly associated with living microorganisms. However, V treatment with E. andrei showed enzyme activities very similar to controls, despite having a stronger reduction of microbial biomass. This may indicate the formation of complexes between enzymes and humic substances during vermicomposting (Benitez et al., 2005) or the presence of a less efficient microbial community for organic matter degradation in controls (Aira et al., 2007b). Although earthworms exerted an important effect on the microbial biomass, the performance of the enzyme activity was determined by both the type of waste used in the vermicomposting and the presence or absence of earthworms. Both treatments were suitable for the reduction and stabilization of microbial biomass of sewage sludge. Earthworm activity had a strong effect on the abundance of PLFAs with a significant reduction in Gram+ bacteria (F1,8 = 134.119, p < 0.0001), Gram bacteria (F1,8 = 106.862, p < 0.0001) and fungi (F1,8 = 76.260, p < 0.0001) with regard to control during vermicomposting (Table 3). Similar results have been reported by Gómez-Brandón et al. (2011a) during vermicomposting of pig slurry with E. fetida, suggesting that this earthworm modified the structure of microbial communities. In the same way, E. andrei affected the abundance of bacterial and fungal PLFAs during vermicomposting of fresh and composted sewage sludge. Previous studies have observed that epigeic earthworms had a greater effect on Gram+ bacteria than Gram bacteria through the gut associated processes and that Gram bacteria can survive the transit through the earthworm gut (Gómez-Brandón et al., 2011c; Williams et al., 2006). In accordance with this, V treatment with earthworms showed a higher reduction in Gram+ bacteria than Gram bacteria compared to controls throughout the process. Likewise, E. andrei had a greater effect on Gram+ bacteria in the

Please cite this article in press as: Villar, I., et al. Changes in microbial dynamics during vermicomposting of fresh and composted sewage sludge. Waste Management (2015), http://dx.doi.org/10.1016/j.wasman.2015.10.011

338.2 ± 15.6 214.7 ± 11.1a 217.8 ± 15.3a 111.1 ± 9.1a 159.9 ± 13.0b 34.5 ± 5.1a 115.5 ± 6.5b 46.4 ± 1.8a 95.0 ± 2.6b 22.5 ± 4.9ac 69.2 ± 3.1b 10.3 ± 1.4a 47.1 ± 3.5b 0.9 ± 0.3a 69.8 ± 8.6b 270.1 ± 16.8 113.7 ± 11.3b 184.6 ± 13.0c 38.4 ± 3.8c 102.3 ± 9.8a 26.6 ± 4.0a 57.8 ± 5.2c 14.3 ± 2.7c 52.0 ± 5.0a 13.5 ± 3.0a 51.3 ± 2.8b 10.5 ± 1.7a 24.0 ± 0.6b 11.0 ± 1.2c 26.5 ± 2.2b

a

178.5 ± 7.2 89.2 ± 10.6b 160.2 ± 5.4c 42.6 ± 3.5c 97.7 ± 4.6a 28.5 ± 1.9a 59.1 ± 4.4c 27.3 ± 4.3c 44.0 ± 5.6ac 23.4 ± 2.6a 53.3 ± 3.1c 17.6 ± 3.6a 27.8 ± 0.7b 12.6 ± 2.1c 16.8 ± 1.0c

197.0 ± 8.8 274.9 ± 12.1a 270.9 ± 16.9a 121.3 ± 7.9a 159.2 ± 10.1b 38.0 ± 4.8a 98.5 ± 2.5b 42.5 ± 1.6a 96.4 ± 9.5b 27.9 ± 2.4a 77.5 ± 2.2b 11.4 ± 1.6a 38.5 ± 4.0b 2.8 ± 0.1a 52.4 ± 5.9b 0 14 28 42 56 70 91 112

In each parameter different letters in the same time of sampling are significantly different (Tukey post hoc test p < 0.05).

370.6 ± 14.9 313.0 ± 14.4a 321.3 ± 13.7a 134.2 ± 7.7a 211.0 ± 13.1b 28.4 ± 4.7a 144.7 ± 10.2b 41.6 ± 1.2a 107.1 ± 1.9b 18.7 ± 3.1a 54.1 ± 4.9b 8.9 ± 1.5a 25.9 ± 2.2b 0.4 ± 0.1a 33.3 ± 3.2b

b a

Control Control E. andrei Control E. andrei

a

E. andrei E. andrei E. andrei

Control CV V CV V

a

CV

E. andrei Control

dw) V

1

Fungi (lg g dw) 1

bacteria (lg g Gram dw) 1

Gram+ bacteria (lg g Time

Table 3 Changes in PLFAs without earthworms (control) and in the presence of E. andrei in vermicomposting (V) and composting–vermicomposting (CV). Values are means ± standard error (n = 3).

186 ± 17.2b 62.5 ± 8.1b 105.8 ± 10.8c 34.6 ± 3.5c 78.4 ± 10.2d 26.4 ± 3.6a 39.0 ± 4.6a 19.4 ± 2.5c 38.7 ± 4.0a 17.0 ± 5.4c 43.2 ± 7.0a 9.6 ± 1.9a 26.4 ± 0.4c 8.1 ± 1.4c 36.6 ± 2.2d

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Control

8

first samplings of CV treatment. Also, a significant decrease in the fungal biomass was observed in the presence of earthworms. This effect on fungal populations was reported by other authors. Huang et al. (2013) and Fernández-Gómez et al. (2013) found a decrease in fungal biomass in the end products after vermicomposting with E. fetida. Such decreases may be due to the fact that earthworms can feed on fungi that provide them an important source of nutrients (Schönholzer et al., 1999). The decrease of microbial groups was greater in V treatment with earthworms (>98.6%) than in CV treatment with earthworms (93% Gram+ and 96% for Gram and fungi), while the reduction in controls was higher in CV treatment for Gram+ bacteria (90.6%) compared to V treatment (73.4%) and similar for Gram bacteria (90–91%) and fungi (79–80%), showing a significant interaction between treatment, time and earthworms presence/absence for all indicators of specific microbial groups: Gram+ (F6,48 = 8.697, p < 0.01), Gram (F6,48 = 6.216, p < 0.01) and fungi (F6,48 = 4,989, p < 0.01). Regarding the last samplings, it was observed that treatments with earthworms, both in V and CV, had a lower concentration of bioindicators of microbial groups than the controls. It has been found that the degradation and mineralisation of organic matter in the controls of vermicomposting is insufficient compared to treatment with earthworms (Elvira et al., 1996). So, the greatest microbial load in controls may indicate the presence of organic matter available for the microorganisms, being lower in CV treatment due to its passage through the thermophilic phase of composting. As can be seen in the last sampling, final products presented different microbial communities. Lores et al. (2006) reported that the fingerprint of the microbial community of a vermicompost depends on the type of substrate and earthworm species used in the process. Fernández-Gómez et al. (2012) reported that vermicomposts produced from wastes of different nature and origin can contain similar microbial communities, if the same earthworm species is used. Sen and Chandra (2009) showed divergent bacterial community in compost and vermicompost obtained from the same initial waste, despite similar changes in their physicochemical parameters during the processes. In this case, the microbial community seemed to differ depending on if V or CV treatment were applied and on the presence or absence of earthworms. Differences may be attributable to the distinct physicochemical and microbiological composition of the waste and the interactions of earthworms on microbial communities. 4. Conclusions In this study, the ability of the earthworm E. andrei to degrade fresh and composted municipal sewage sludge was shown. The PLFA analysis indicated that E. andrei reduced microbial biomass, both bacterial and fungal, and that the composition of the microbial community depended on the presence or absence of earthworms as well as the treatment used. Earthworm incorporation after the thermophilic stage of composting accelerated the degradation of the waste, with a greater decrease in the phosphorus and carbon cycling enzymes. Therefore, the combined compost ing–vermicomposting process can be a good alternative to the management of sewage sludge. Acknowledgments This study was financially supported by the Xunta de Galicia (Regional Autonomous Government of Galicia, Spain) (09MDS024310PR). The authors thank the wastewater treatment plant of Cangas for supplying sewage sludge and the research support services of the University of Vigo (CACTI) for the carbon and nitrogen analysis. The authors also thank Emilio Rodríguez Cochón

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